Hybridomas synthesizing monoclonal antibodies to Bordetella pertussis toxins

Hybridomas synthetizing monoclonal antibodies (McAb) to B. pertussis toxin (BPT) and endotoxin, or lipopolysaccharide (LPS), were obtained. The specificity of McAb to BPT was confirmed in the leukocytosis-stimulating factor neutralization test. Two hybridomas synthetized McAb, seemingly active against the common determinant of BPT and LPS. The McAb of one hybridoma reacted with the crude extract of BPT.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus

A series of hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus was obtained and their cultural properties were characterized. HEMA-12 and HEMA-24 secreted monoclonal IgG2b antibodies, HEMA-101 secreted monoclonal IgG1 antibodies, HEMA-31, HEMA-9 and HEMA-11 secreted monoclonal IgG2 antibodies. According to the results of the indirect immunofluorescence test, the titer of specific immunoglobulins in the culture fluid was 1:16-1:32, but sometimes reached 1:64-1:128. The titer of antibodies in ascitic fluid amounted to dilutions of 10(4)-10(5). Hybridomas were cloned by the method of ultimate dilutions. The injection of 5-15 million HEMA cells into the abdominal cavity of BALB/c mice induced the formation of ascitic tumors in the animals within 7-11 days and the accumulation of ascitic fluid in a volume of 1-4 ml. Hybridomas, found to be capable of passage from mouse to mouse, underwent 5-16 passages during the term of observation.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Two surface antigens expressed on proliferating mouse T lymphocytes defined by rat monoclonal antibodies

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating, as opposed to resting, mouse T cells. In this report, the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells, some bone marrow cells, and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes, spleen cells, bone marrow cells, or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It, too, appears to be a dimer, with a m.w. of 95,000 daltons under nonreducing conditions, decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known, its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation.     

Production and characterization of monoclonal antibodies to Aeromonas sobria surface antigens

A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria.     

Production and characterization of monoclonal antibodies to Aeromanas sobria surface antigens

Abstract A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria .     

The detection of two surface antigens on human lung macrophages using monoclonal antibodies

Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.     

Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.     

Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus

Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein-Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen.     

Binding of monoclonal antibodies to cerebral cytoplasmic tetrodotoxin-sensitive protein to human lymphocytes

To examine whether lymphocytes express antigenic determinants of brain cytoplasmic tetrodotoxin-sensitive protein (CTSP), anti-CTSP monoclonal antibody (Bab) binding to human peripheral blood lymphocytes was studied using ELISA assay. It was shown that the Mab bound human lymphocytes in a dose-dependent fashion. Halph-maximal binding of this Mab was significantly enhanced if test-cells were pretreated with concanavalin A or with mixed lymphocyte culture supernatant. Results are discussed from the point of view of the hypothesis that CTSP are metabolic precursors of membrane sodium channels.     

Obtaining monoclonal antibodies to Bordetella pertussis antigens

The immunization of mice with the protein fraction of B. pertussis strain 305 has made it possible to obtain hybridomas producing monoclonal antibodies to B. pertussis antigens. Ascitic fluids containing monoclonal antibodies react in the ELISA in high titers and actively agglutinate B. pertussis strains 305 and 475.     

Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Stimulation of human T lymphocytes by PHA-activated autologous T lymphocytes: Analysis of the role of Ia-like antigens with monoclonal antibodies

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Production of hybridomas synthesizing monoclonal antibodies to streptococcal group A polysaccharide

Nine stable hybridomas synthetizing monoclonal antibodies to the antigenic determinants of polysaccharide A of group A streptococcus were obtained. Three monoclonal antibodies possessed precipitating properties. The formation of hybridomas was found to be influenced by the presence of immune splenocytes and the standard conditions of cell fusion. The highest yield of hybridomas was observed under the conditions ensuring the growth of cell in 80-100% of the wells. Rapid and specific screening was found to be an important stage in obtaining hybridomas.